A: Transfer a quantity of finely powdered Tablets,equivalent to about 200mg of leucovorin calcium,to a conical flask,add 10mLof water,shake vigorously,sonicate for 10minutes,and filter.Transfer the filtrate to a stoppered centrifuge tube,add about 125mg of ammonium oxalate,shake vigorously,and centrifuge until a clear supernatant is obtained.Transfer the supernatant to another stoppered centrifuge tube,add 1mLof methanol and 3drops of hydrochloric acid,and shake vigorously.If the preparation is cloudy,add methanol until a clear solution is obtained,and filter if necessary to remove any undissolved material.Cool the preparation at 0until a precipitate forms,and centrifuge for 1to 2minutes.[NOTE—The cooling and centrifuging steps may be repeated if necessary to increase the amount of precipitate collected.]Decant the supernatant,add 2mLof methanol to the tube,shake vigorously to dissolve the precipitate,and transfer the contents to a beaker.Evaporate under a current of air to dryness,and dry the residue at 50for 30minutes:the IRabsorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Leucovorin Calcium RS.

B: The retention time of the leucovorin peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,as obtained in the Assay.

Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Procedure— Determine the amount of C20H21CaN7O7dissolved from UVabsorbances at the wavelength of maximum absorbance at about 284nm using filtered portions of the solution under test,diluted with water if necessary,in comparison with a Standard solution having a known concentration of USP Leucovorin Calcium RSin the same Medium.

Tolerances— Not less than 75%(Q)of the labeled amount of C20H21CaN7O7is dissolved in 30minutes.

Procedure for content uniformity— Using individual intact Tablets,prepare solutions in water having known concentrations of about 10µg of leucovorin calcium per mL.Prepare a Standard solution of USP Leucovorin Calcium RSin water having a known concentration of about 10µg per mL.Concomitantly determine the absorbances of the test solutions and the Standard solution in 1-cm cells at the wavelength of maximum absorbance at about 284nm,with a suitable spectrophotometer,using water as the blank.Calculate the quantity,in mg,of C20H21CaN7O7in each Tablet taken by the formula: in which Tis the labeled quantity,in mg,of leucovorin calcium in the Tablet;Dis the concentration,in µg per mL,of the solution from the Tablet,based on the labeled quantity per Tablet and the extent of dilution;Cis the concentration,in µg per mL,of USP Leucovorin Calcium RSin the Standard solution;and AUand ASare the absorbances of the solution from the Tablet and the Standard solution,respectively.

100(ri/rt),

Diluting solvent— Prepare a mixture of water and methanol (80:20).

Mobile phase— Prepare a 0.005Msolution of tetrabutylammonium phosphate in Diluting solvent.Adjust the pHof this solution to 7.5by the addition of 50%(w/v)sodium hydroxide solution,filter,and degas.

Standard preparation— Dissolve accurately weighed quantities of USP Leucovorin Calcium RSand USP10-Formylfolic Acid RSin water to obtain a solution having known concentrations of about 500µg of USP Leucovorin Calcium RSand 10µg of USP10-Formylfolic Acid RSper mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 50mg of leucovorin,to a 100-mLvolumetric flask.Add about 50mLof water,sonicate for 30minutes,dilute with water to volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 2.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between leucovorin and 10-formylfolic acid is not less than 1.5;and the relative standard deviation for replicate injections,and retention times is not more than 2.0%for the leucovorin peak.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak area responses.The relative retention times are about 2.3for 10-formylfolic acid and 1.0for leucovorin.Calculate the quantity,in mg,of C20H23N7O7in the portion of Tablets taken by the formula: in which 473.45and 511.50are molecular weights of leucovorin and anhydrous leucovorin calcium,respectively;Lis the labeled amount,in mg,of leucovorin in each Tablet;Dis the concentration,in mg per mL,of leucovorin in the Assay preparation,based on the labeled quantity per Tablet and the extent of dilution;Cis the concentration,in mg per mL,of anhydrous USP Leucovorin Calcium RSin the Standard preparation;and rUand rSare the peak area responses obtained from the Assay preparationand the Standard preparation,respectively.